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WO2007047604A2 - Inhibition de sirt1 - Google Patents

Inhibition de sirt1 Download PDF

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Publication number
WO2007047604A2
WO2007047604A2 PCT/US2006/040426 US2006040426W WO2007047604A2 WO 2007047604 A2 WO2007047604 A2 WO 2007047604A2 US 2006040426 W US2006040426 W US 2006040426W WO 2007047604 A2 WO2007047604 A2 WO 2007047604A2
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WIPO (PCT)
Prior art keywords
cells
alkyl
aminocarbonyl
aryl
sirtl
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PCT/US2006/040426
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WO2007047604A3 (fr
Inventor
Peter Distefano
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Elixir Pharmaceuticals Inc
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Elixir Pharmaceuticals Inc
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Priority to US12/090,167 priority Critical patent/US20090022694A1/en
Publication of WO2007047604A2 publication Critical patent/WO2007047604A2/fr
Publication of WO2007047604A3 publication Critical patent/WO2007047604A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • a SIRTl inhibitor can be used to enhance the properties of cells, particularly cultured cells and cells for transplantation, hi one aspect, this disclosure features a method of modulating cells, e.g., progenitor cells or other cells described herein.
  • the method includes inhibiting SIRTl activity in cells, e.g., in vitro.
  • the method can include culturing cells, e.g., in tissue culture.
  • the cells are generally mammalian cells, e.g., mammalian cells that express SIRTl, e.g., human cells.
  • the method can further include maintaining the cells in vitro in the presence of an effective amount of a SIRTl inhibitor, e.g., a small molecule inhibitor of SIRTl (e.g., an inhibitor described herein), particularly an inhibitor of a SIRTl enzymatic activity, e.g., deacetylase activity.
  • a SIRTl inhibitor e.g., a small molecule inhibitor of SIRTl (e.g., an inhibitor described herein), particularly an inhibitor of a SIRTl enzymatic activity, e.g., deacetylase activity.
  • the SIRTl inhibitor has an IC50 of 10 ⁇ M, 1 ⁇ M, 100 nM, 50 nM, 10 nM or less.
  • the maintaining can include combining a culture that includes the cells with a substantially pure preparation of the SIRTl inhibitor.
  • the cells can be obtained directly from a subject, can be cells of a primary cell line, or an immortalized cell line. In one embodiment, the cells are not terminally differentiated. In one embodiment, the cells are not transformed (with respect oncogenesis).
  • the cells can be in the form of isolated cells (e.g., in a sheet or individually in solution or on a substrate), tissue, or an organ, or parts thereof.
  • the SIRTl inhibitor can be provided in an amount effective to prolong lifespan of the cells or to increase the replicative capacity of the cells.
  • the term "prolonged lifespan” refers to an increase in the time until a cell terminally differentiates or stops proliferating.
  • the term “replicative capacity” refers to the number of times a cell can divide, e.g., the number of cell divisions until the cell terminally differentiates, stops replicating, senesces, or dies. These parameters can be evaluated by assaying a culture that contains the inhibitor and a control culture to determine the time until terminal differentiation or the end of proliferations, and so forth.
  • the SIRTl inhibitor can decrease oxidative damage (e.g., and thereby increase cell survival) associated with a disease (e.g., liver disease) or procedure (e.g., a surgical procedure).
  • a disease e.g., liver disease
  • procedure e.g., a surgical procedure.
  • the cells can be cells that respond to a SIRTl inhibitor by having increased resistance to oxidative stress.
  • An effective amount can be determined by evaluating a range of concentrations of the SIRTl inhibitor, e.g., to identify one or more concentrations that produce a statistically significant effect.
  • An effective amount of a SIRTl inhibitor, for example, a SIRTl inhibitor described herein may range, for example, from a concentration about 0.01-10 times the IC50 of the particular compound, e.g., from 0.1 to 2 times the IC50 when used in culture. Effective doses will also vary depending on the specific culture conditions and cell type, as well as the possibility of co-usage with other agents.
  • the cells are transplantable, e.g., they can be administered to a subject, e.g., a subject that was the source of the cells, or a different subject, e.g., a immunosuppressed subject, or a normal subject.
  • a subject e.g., a subject that was the source of the cells
  • a different subject e.g., a immunosuppressed subject
  • the cells can be a form suitable for transplantation, e.g., organ transplantation.
  • the method can further include administering the cells to a subject in need thereof, e.g., a mammalian subject, e.g., a human subject.
  • the source of the cells can be a mammal, preferably a human.
  • the source or recipient of the cells can also be a non- human subject, e.g., an animal model.
  • the term "mammal” includes organisms, which include mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans.
  • transplantable cells can be obtained from any of these organisms, including a non-human transgenic organism.
  • the transplantable cells are genetically engineered, e.g., the cells include an exogenous gene or have been genetically engineered to inactivate or alter an endogenous gene.
  • the cells can be, for example, administered to a subject who has experienced or is at risk of experiencing senescence (e.g., abnormal senescence), diabetes (e.g., type I or II), metabolic syndrome, skeletal muscle disease (e.g., Duchene muscular dystrophy,
  • a chronically degenerative disease such as cardiac muscle disease, neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, Huntington's Disease), bone disease (e.
  • the method further includes contacting a test agent to the cells cultured with the SIRTl inhibitor.
  • the contacting can be used to characterize the test agent, e.g., a drug or drug candidate, e.g., to evaluate toxicity, a biochemical property, and responsiveness of the cell.
  • the disclosure features a cell culture medium that includes a buffered medium, growth factors, and a SIRTl inhibitor, e.g., an inhibitor having an IC50 for SIRTl enzymatic activity of less than 10 ⁇ M, e.g., an inhibitor described herein.
  • the disclosure also features a cell culture that includes mammalian cells (e.g., stem cells, sperm, or eggs) and a SIRTl inhibitor, and optionally a medium that comprises nutrients and growth factors.
  • the medium further includes a cryoprotectant, adjuvant, anti-oxidant, and so forth.
  • a method of culturing cells includes inhibiting SIRTl activity in cells in vitro.
  • the method includes maintaining the cells in vitro in the presence of an effective amount of a SIRTl inhibitor (e.g., a small molecule). In some embodiments, the method includes combining the cells with a substantially pure preparation of the SIRTl inhibitor. In some embodiments, the SIRTl inhibitor has an IC50 of 10 ⁇ M or less.
  • a SIRTl inhibitor e.g., a small molecule
  • the method the cells are mammalian (e.g., human). In some embodiments, the cells are not terminally differentiated. In other embodiments, prior to culturing, the cells are senescent or terminally differentiated. In a preferred embodiment, prior to culturing, the cells are senescent. In another preferred embodiment, prior to culturing, the cells are terminally differentiated. In some embodiments, the cells are not transformed. In some embodiments, the SERTl inhibitor prolongs lifespan of the cells. In other embodiments, wherein replicative capacity of the cells is increased. In some embodiments, the cells are transplantable. The cells may be genetically engineered. In some embodiments, the method also includes administering the cells (e.g., genetically engineered cells) to a subject in need thereof.
  • the cells are bone marrow cells, cardiac muscle cells, dopamine-producing cells, osteoblasts, osteocytes, hepatocytes, stromal cells, fetal brain cells, pancreatic B cells, or myoblasts.
  • the cells are cardiac muscle cells, dopamine-producing cells, osteoblasts, osteocytes, hepatocytes, fetal brain cells, pancreatic B cells, or myoblasts.
  • the cells are cardiac muscle cells.
  • the cells are dopamine-producing cells.
  • the cells are osteoblasts.
  • the cells are osteocytes.
  • the cells are hepatocytes.
  • the cells are fetal brain cells.
  • the cells are pancreatic B cells.
  • the cells are myoblasts.
  • the cells are stem cells.
  • the stem cell is committed to a mesenchymal, hematopoietic, adipogenic, hepatogenic, neurogenic, gliogenic, chondrogenic, vasogenic, myogenic, chondrogenic, or osteogenic lineage.
  • the stem cell is committed to an adipogenic, hepatogenic, neurogenic, gliogenic, vasogenic, myogenic, or osteogenic lineage.
  • the stem cell is committed to an adipogenic lineage.
  • the stem cell is committed to a hepatogenic lineage.
  • the stem cell is committed to a neurogenic lineage.
  • the stem cell is committed to a gliogenic lineage. In a more preferred embodiment, the stem cell is committed to a vasogenic lineage. In a more preferred embodiment, the stem cell is committed to a myogenic lineage. In a more preferred embodiment, the stem cell is committed to an osteogenic lineage.
  • the cells are administered to a subject who has experienced or is at risk of experiencing abnormal senescence, diabetes (e.g., type I or II), metabolic syndrome, skeletal muscle disease (e.g., Duchene muscular dystrophy, Becker's dystrophy, or myotonic dystrophy), ALS under neurodegenerative disease, spinal cord trauma, heart disease, stroke, macular degeneration, a chronically degenerative disease (such as cardiac muscle disease, neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, or Huntington's Disease), bone disease (e.g., osteoporosis), a blood disease (e.g., a leukemia) or liver disease (e.g., due to alcohol abuse or hepatitis)), or other condition characterized by unwanted cell loss, or a subject who has undergone chemotherapy or radiation treatment, a subject that has suffered a wound (e.g., a surgical wound), a burn, an ulcer (e.g., ulcer in a diabetic,
  • a wound
  • the cells are administered to a subject who has experienced or is at risk of experiencing abnormal senescence, diabetes (e.g., type I or II), metabolic syndrome, skeletal muscle disease (e.g., Duchene muscular dystrophy, Becker's dystrophy, or myotonic dystrophy), ALS under neurodegenerative disease, spinal cord trauma, heart disease, stroke, macular degeneration, a chronically degenerative disease (such as cardiac muscle disease, neurodegenerative disease (e.g.,
  • the cells are administered to a subject who has experienced or is at risk of experiencing abnormal senescence.
  • the cells are administered to a subject who has experienced or is at risk of experiencing diabetes (e.g., type I or II).
  • the cells are administered to a subject who has experienced or is at risk of experiencing metabolic syndrome.
  • the cells are administered to a subject who has experienced or is at risk of experiencing skeletal muscle disease (e.g., Duchene muscular dystrophy, Becker's dystrophy, or myotonic dystrophy).
  • skeletal muscle disease e.g., Duchene muscular dystrophy, Becker's dystrophy, or myotonic dystrophy.
  • the cells are administered to a subject who has experienced or is at risk of experiencing ALS under neurodegenerative disease.
  • the cells are administered to a subject who has experienced or is at risk of experiencing spinal cord trauma.
  • the cells are administered to a subject who has experienced or is at risk of experiencing heart disease.
  • the cells are administered to a subject who has experienced or is at risk of experiencing stroke.
  • the cells are administered to a subject who has experienced or is at risk of experiencing macular degeneration.
  • the cells are administered to a subject who has experienced or is at risk of a chronically degenerative disease (such as cardiac muscle disease, neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, or Huntington's Disease), hi a more preferred embodiment, the cells are administered to a subject who has experienced or is at risk of experiencing liver disease (e.g., due to alcohol abuse or hepatitis), hi a more preferred embodiment, the cells are administered to a subject who has experienced or is at risk of experiencing a condition characterized by unwanted cell loss, hi a more preferred embodiment, the cells are administered to a subject who has suffered or is at risk of suffering with an ulcer (e.g., ulcer in a diabetic, e.g., diabetic foot ulcer), hi a more preferred embodiment, the cells are administered to a subject who has experienced or is at risk of experiencing a sore.
  • a chronically degenerative disease such as cardiac muscle disease, neuro
  • the method also includes evaluating one or more test compounds by contacting the test compound to the cells.
  • the SIRTl inhibitor comprises a compound having formula (I):
  • R 1 and R 2 together with the carbons to which they are attached, form Cs-C 10 cycloalkyl, C 5 -C 10 heterocyclyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, or C 6 -C 10 heteroaryl, each of which maybe optionally substituted with 1-5 R 5 ; or R 1 is H, S-alkyl, or S-aryl, and R 2 is amidoalkyl wherein the nitrogen is substituted with alkyl, aryl, or arylalkyl, each of which is optionally further substituted with alkyl, halo, hydroxy, or alkoxy; R 3 and R 4 , together with the carbons to which they are attached, form C 5 -C 10 cycloalkyl, C 5 -C 10 heterocyclyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C
  • R 1 and R 2 together with the carbons to which they are attached, form C 5 -C 10 cycloalkyl, C 5 -C 10 heterocyclyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, or C 6 -C 10 heteroaryl, each of which maybe optionally substituted with 1-5 R 5 .
  • R and R together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl.
  • R 1 and R 2 are substituted with R 5 .
  • R 3 and R 4 together with the carbons to which they are attached, form C 6 -C 10 aryl. In some embodiments, R 3 and R 4 are substituted with R 6 . hi some embodiments, R 6 is halo or C 1 -C 6 alkyl.
  • n 0.
  • X is NR 7 .
  • n 0 and X is NR 7 .
  • the compound of formula (I) compound has the formula
  • R 6 is halo or C 1 -C 6 alkyl. In some embodiments, R 5 is aminocarbonyl.
  • the compound of formula (X) has the formula (XI) below:
  • R 6 is halo or alkyl.
  • R 5 is aminocarbonyl
  • R 6 is halo or alkyl and wherein R 5 is aminocarbonyl.
  • the compound of formula (X) is 6-Chloro-2,3,4,9- tetrahydro-lH-carbazole-1-carboxylic acid amide.
  • the compound can be a compound of formula (IV) having a high enantiomeric excess of a single isomer, wherein the optical rotation of the predominant isomer is negative.
  • a compound of formula (IV), (V), or (VII) is administered having a high enantiomeric excess of a single isomer, where the predominant isomer has the same absolute configuration as the negative isomer of the compound of formula (VI) as corresponds to the asterisk carbon shown above.
  • the SIRTl inhibitor includes a compound having formula (XXII):
  • R 6 is chloro or methyl.
  • p is 1.
  • the SIRTl inhibitor includes a compound having formula (XXIII): formula (XXIII)
  • R 1 is H, halo, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl; or when taken together with R 2 and the carbon to which it is attached, forms C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, or C 6 -C 10 heteroaryl; each of which can be optionally substituted with 1-5 R 5 ; R 2 is H, halo, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 6
  • R 8 is H, C 1 -C 6 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 arylalkyl, C 7 -C 12 heteroarylalkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 - C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 7 -C 12 heterocyclylalkyl, C 7 -C 12 cyloalkylalkyl, C 7 -C 12 heterocycloalkenylalkyl, or C 7 -C 12 cycloalkenylalkyl;
  • R 9 is H or C 1 -C 6 alkyl; and each R 10 is independently halo, hydroxy, alkoxy, alkyl, alkenyl, alkynl, nitro, amino, cyano, amido, or aminocarbonyl.
  • R 1 and R 2 taken together, with the carbons to which they are attached, form C 5 -C 1O cycloalkenyl, C 5 -C 1O heterocycloalkenyl, C 6 -C 10 aryl, or C 6 -C 10 heteroaryl.
  • R 1 and R 2 taken together, with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl.
  • R 1 and R 2 taken together, with the carbons to which they are attached, form C 5 -C 1O cycloalkenyl, optionally substituted with 1 or 2 C 1 -C 6 alkyl. In some preferred embodiments, R 1 and R 2 , taken together form a C 5 -C 7 cycloalkenyl ring substituted with C 1 -C 6 alkyl.
  • R 1 is C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 heterocyclyl, C 5 -C 10 cycloalkenyl, or C 5 -C 10 heterocycloalkenyl. In some preferred embodiments, R 1 is C 6 -Ci 0 aryl.
  • R 2 is H, halo, C 1 -C 10 alkyl, or C 1 -C 6 haloalkyl.
  • R 3 is carboxy, cyano, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -C 10 alkoxycarbonyl, C 1 -C 10 alkylthioylcarbonyl, hydrazinocarbonyl, C 1 -C 6 alkylhydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, or hydroxyaminocarbonyl.
  • R 3 is aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, or hydroxyaminocarbonyl. In some preferred embodiments, R 3 is aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, or C 1 -C 6 dialkyl aminocarbonyl.
  • R 3 is H, thioalkoxy or thioaryloxy.
  • R 4 is nitro, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, or amido. In some preferred embodiments, R 4 is amino or amido.
  • R 4 is aminocarbonylalkyl.
  • amino of the aminocarbonylalkyl is substituted with aryl, arylalkyl, alkyl, etc.
  • each substituent can independently be further substituted with halo, hydroxy, or alkoxy.
  • R 3 is aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, or C 1 -C 6 dialkyl aminocarbonyl; and R 4 is amino, C 1 -C 6 alkyl amino C 1 -C 6 dialkyl amino or amido.
  • X is S. In some embodiments, X is NR 8 . In some preferred embodiments, R 8 is H, C 1 -C 6 alkyl or C 7 -C 10 arylalkyl.
  • R 1 is C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 heterocyclyl, C 5 -C 10 cycloalkenyl, or C 5 -C 10 heterocycloalkenyl; or when taken together with R 2 and the carbon to which it is attached, forms C 5 -C 10 cycloalkenyl;
  • R 2 is H, halo, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl; or when taken together with R 1 and the carbon to which it is attached, forms C 5 -C 10 cycloalkenyl;
  • R is aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, or hydroxyaminocarbonyl;
  • R 4 is amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, or amido; and X is S.
  • R 1 and R 2 taken together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl
  • R 3 is aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, or C 1 -C 6 dialkyl aminocarbonyl;
  • R 4 is amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, or amido; and
  • X is S.
  • the SIRTl inhibitor includes a compound having formula (II):
  • R 11 is H, halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 -C 6 haloalkoxy, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 - C 10 heterocycloalkenyl, carboxy, carboxylate, cyano, nitro, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, mercapto, thioalkoxy, thioaryloxy, thioheteroaryloxy, SO 3 (R 13 ), sul
  • R 12 is H, halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 -C 6 haloalkoxy, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 - C 10 heterocycloalkenyl, C 6 -C 10 aryloxy, C 5 -Ci 0 heteroaryloxy, carboxy, carboxylate, cyano, nitro, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, mercapto, thioalkoxy, thio
  • R 14 is hydroxy, carboxy, carboxylate, cyano, nitro, amino, C 1 -C 6 alkyl amino, Ci- C 6 dialkyl amino, oxo, mercapto, thioalkoxy, thioaryloxy, thioheteroaryloxy, SO 3 H, sulfate, S(O)NH 2 , S(O) 2 NH 2 , phosphate, acyl, amidyl, aminocarbonyl, Ci-C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -Ci 0 alkoxycarbonyl, C 1 -C 10 thioalkoxycarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, hydroxyaminocarbonyl, or alkoxyaminocarbonyl;
  • R 15 is halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -Ci 0 alkoxy, Ci-C 6 haloalkoxy, C 6 -C 10 aryloxy, C 5 -Ci 0 heteroaryloxy, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -Ci 2 aralkyl, C 7 -Cn heteroaralkyl, C 3 -C 8 heterocyclyl, C 2 -Ci 2 alkenyl, C 2 -C 12 alkynyl, C 5 -Ci 0 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 arylalkoxy, or C 5 -C 10 heteroarylalkoxy;
  • Z is NR 16 , O, or S; each Y is independently N or CR 18 ;
  • R 16 is H, Ci-Cio alkyl, C 1 -C 6 haloalkyl, C 6 -C 10 aryl, C 5 -Ci 0 heteroaryl, C 7 -Ci 2 aralkyl, C 7 -Ci 2 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 5 -Ci 0 cycloalkenyl, C 5 -Ci 0 heterocycloalkenyl, C 2 -C 12 alkenyl, C 2 -Ci 2 alkynyl; or one of R 11 or R 12 and R 16 form a cyclic moiety containing 4-6 carbons, 1-3 nitrogens, 0-2 oxygens and 0-2 sulfurs; wherein each is optionally substituted with R 17 ;
  • R 17 is halo, hydroxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, Ci-C 6 alkoxy, C 1 -C 6 haloalkoxy, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, oxo, mercapto, thioalkoxy, SO 3 H, sulfate, S(O)NH 2 , S(O) 2 NH 2 , phosphate, acyl, amido, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 thioalkoxycarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, hydroxyaminocarbonyl, or alkoxya
  • Z is NR 16 .
  • Z is NR 16
  • R 16 is C 1 -C 10 alkyl, cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, or C 7 -C 12 heteroaralkyl.
  • R 16 is C 1 -C 10 alkyl, C 6 -C 1O aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, or C 7 -C 12 heteroaralkyl, substituted with one or more halo, alkyl, or alkoxy.
  • R 11 is mercapto, thioalkoxy, thioaryloxy, thioheteroaryloxy, SO 3 (R 13 ), sulfate, S(O)N(R 13 ) 2 , S(O) 2 N(R 13 ) 2 .
  • R 11 is thioalkoxy, thioaryloxy, thioheteroaryloxy.
  • R 11 is thioalkoxy, thioaryloxy, thioheteroaryloxy; substituted with one or more acyl, amido aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -C 1O alkoxycarbonyl, C 1 -C 10 thioalkoxycarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, hydroxyaminocarbonyl, or alkoxyaminocarbonyl.
  • R 11 is thioalkoxy substituted with one or more amido, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, or C 1 -C 6 dialkyl aminocarbonyl. In more preferred embodiments, R 11 is thioalkoxy substituted with aminocarbonyl.
  • R 12 is C 1 -C 10 alkyl, C 6 -C 1 O aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl.
  • R 12 is C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, or C 7 -C 12 heteroaralkyl.
  • R 12 is C 1 -C 10 alkyl substituted with one or more halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 6 -C 10 aryloxy, or C 5 -C 10 heteroaryloxy. In more preferred embodiments, R 12 is C 1 -C 10 alkyl substituted with aryloxy. In some embodiments, each Y is N.
  • R ⁇ is thioalkoxy, thioaryloxy, thioheteroaryloxy; substituted with one or more acyl, amido aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 - C 6 dialkyl aminocarbonyl, C 1 -C 10 alkoxycarbonyl, C 1 -C 10 thioalkoxycarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 6 dialkyl hydrazinocarbonyl, hydroxyaminocarbonyl, or alkoxyaminocarbonyl;
  • R 12 is C 1 -C 10 alkyl substituted with one or more halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 6 -C 10 aryloxy, or C 5 -C 10 heteroaryloxy;
  • Z is
  • the SIRTl inhibitor includes a compound having formula (III):
  • R 21 is halo, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl; or when taken together with R 22 and the carbon to which it is attached, forms C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 1O aryl, or C 5 -C 10 heteroaryl; each of which can be optionally substituted with 1-5 R 25 ; R 22 is halo, C 1 -C 10 alkyl, C 1 -C
  • R 23 is H, halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 1O cycloalkenyl, C 5 -C 10 heterocycloalkenyl, carboxy, carboxylate, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, acyl, C 1 -C 10 alkoxycarbonyl, C 1 -C 10 thioalkoxycarbonyl;
  • R 24 is, halo, hydroxy, Ci-C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 -C 6 haloalkoxy, C 6 -Ci 0 aryl, C 5 -Ci 0 heteroaryl, C 7 -C 12 aralkyl, C 7 -Ci 2 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 1O cycloalkenyl, C 5 - C 10 heterocycloalkenyl, C 6 -C 10 aryloxy, C 5 -C 1O heteroaryloxy, carboxy, carboxylate, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, mercapto, thioalkoxy, thioaryloxy,
  • R 28 is H, C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 - C 12 heteroaralkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, or C 5 -C 10 cycloalkenyl;
  • Q is S, O, or NR 29 ;
  • R 29 is H, C 1 -C 6 alkyl, C 7 -C 12 aralkyl, or C 7 -C 12 heteroaralkyl;
  • P is N or CR 30 ; and R 30 is H or C 1 -C 6 alkyl.
  • R 21 and R 22 together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, or C 5 -C 10 heteroaryl. In some preferred embodiments, R 21 and R 22 , together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl.
  • R 23 is hydroxy, C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 1O cycloalkenyl, C 5 -C 10 heterocycloalkenyl, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, or acyl.
  • R 23 is C 3 -C 8 cycloalkyl, C 5 -C 8 heterocyclyl, C 5 -C 1O cycloalkenyl, or C 5 -C 10 heterocycloalkenyl.
  • R 24 is halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 -C 6 haloalkoxy, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryloxy, C 5 -C 10 heteroaryloxy, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, mercapto, thioalkoxy, thioaryloxy, or thioheteroaryloxy.
  • R 24 is C 1 -C 10 alkyl, thioalkoxy, thioaryloxy, or thioheteroaryloxy. In preferred embodiments, R 24 is C 1 -C 10 alkyl or thioalkoxy; and R 27 is carboxy, carboxylate, cyano, nitro, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, SO 3 H, sulfate ⁇ S(O)N(R 28 ) 2 , S(O) 2 N(R 28 ) 2 , phosphate, acyl, amidyl, aminocarbonyl, C 1 - C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -C 10 alkoxycarbonyl, C 1 -C 1O thioalkoxycarbonyl, hydrazinocarbonyl, C 1 -C 6 alkyl hydrazinocarbonyl, C 1 -C 10 alkyl
  • Y is N.
  • R 21 and R 22 together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, or C 5 -C 10 heteroaryl;
  • R 23 is hydroxy, C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, amino, C 1 -C 6 alkyl amino, C 1 - C 6 dialkyl amino, or acyl;
  • R 24 is C 1 -C 10 alkyl, thioalk
  • R 21 and R together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl, or C 5 -C 10 heterocycloalkenyl;
  • R 23 is C 1 -C 10 alkyl, C 7 - C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 1O heterocycloalkenyl, amino, C 1 -C 6 alkyl amino, or C 1 -C 6 dialkyl amino;
  • R 24 is C 1 -C 10 alkyl, thioalkoxy, thioaryloxy, or thioheteroaryloxy;
  • R 27 is carboxy, carboxylate, SO 3 H, sulfate, S(O)N(R
  • the SIRTl inhibitor includes a compound having formula (IV):
  • R 41 is H, halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 - C 6 haloalkoxy, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 - C 10 heterocycloalkenyl, carboxy, carboxylate, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, acyl, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -C 10 alkoxy
  • R 42 and R 43 together with the carbons to which they are attached, form C 5 -C 10 cycloalkyl, C 5 -C 10 heterocyclyl, C 5 -C 1O cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 10 aryl, or C 6 -C 10 heteroaryl, each of which is optionally substituted with 1-4 R 45 ; or R 44 is H, halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 - C 6 haloalkoxy, C 6 -C 10 aryl, C 5 -C 1O heteroaryl, C 7 -C 12 aralkyl, C 7 -C 12 heteroaralkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkyl
  • R 45 is halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 -C 6 haloalkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, oxo, carboxy, carboxylate, cyano, nitro, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, mercapto, thioalkoxy, thioaryloxy, thioheteroaryloxy, SO 3 H, sulfate, S(O)N(R 46 ) 2 , S(O) 2 N(R 46 ) 2 , phosphate, C 1 -C 4 alkylenedioxy, acyl, amido, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, C 1 -C 10 alkoxy
  • R 46 is H, C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 7 -C 12 aralkyl, C 7 - C 12 heteroaralkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, or C 5 -C 10 cycloalkenyl; and
  • M is NR 47 , S, or O
  • R 47 is H, halo, hydroxy, C 1 -C 10 alkyl, C 1 -C 6 haloalkyl, C 1 -C 10 alkoxy, C 1 - C 6 haloalkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, carboxy, carboxylate, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, acyl, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, or C 1 -C 10 alkoxycarbonyl.
  • R 42 and R 43 together with the carbons to which they are attached, form C 6 -C 10 aryl, or C 6 -C 10 heteroaryl. In some preferred embodiments, R 42 and R 43 , together with the carbons to which they are attached, form phenyl. In more preferred embodiments, R 42 and R 43 , together with the carbons to which they are attached, form phenyl; and are substituted with halo or C 1 -C 10 alkyl.
  • R 41 is C 1 -C 10 alkyl; and R 44 is H, halo, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocyclyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, acyl, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, amido, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, carboxy, or C 1 -C 10 alkoxycarbonyl.
  • R 41 is C 1 -Ci 0 alkyl
  • R 44 is acyl, amino, C 1 -C 6 alkyl amino, C 1 -C 6 dialkyl amino, amido, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl, C 1 -C 6 dialkyl aminocarbonyl, carboxy, or C 1 -C 10 alkoxycarbonyl
  • R 42 and R 43 together with the carbons to which they are attached, form C 6 -C 10 aryl, or C 6 -C 10 heteroaryl
  • M is O.
  • the SIRTl inhibitor includes a compound having formula V:
  • X is a member selected from the group consisting of O and S
  • L 1 and L 2 each represent members independently selected from the group consisting of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of L 1 and L 2 • represents a member selected from the group consisting of O and S
  • each instance of R and of L 1 and L 2 independently represents a member selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl and-CO 2 R 4
  • R 1 and R 2 each represent members independently selected from the group consisting of hydrogen, C 1-6 alkoxy, C 0-6 alkoxy-aryl and hydroxyl
  • R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, aryl,-OR 4 ,- NR 4 R 4 , -CO 2 R 4 , -C(O) R 4 , -C(O)NR 4 R 4 , -CN, -NO 2 and halogen
  • R 4 independently is selected from
  • X is a member selected from the group consisting of O and S
  • L 1 and L 2 each represent members independently selected from the group consisting of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of L 1 and L 2 represents a member selected from the group consisting of O and S; each instance of R and of L 1 and L 2 independently represents a member selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl and-CO 2 R 4 ;
  • R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, aryl,-OR 4 ,-NR 4 R 4 , -CO 2 R 4 , -C(O) R 4 , -C(O)NR 4 R 4 , -CN, -NO 2 and halogen;
  • R 4 independently is selected from the group consisting of hydrogen and C 1- 6 alkyl; R 1 and R 2 taken together with the carbons to which they are attached form a six- membered lactone ring
  • the compound has the following structure:
  • R 1 is a member selected from the group consisting of hydrogen, C 1 . ealkoxy and C 0-6 alkoxy-aryl ;
  • R 2 is selected from the group consisting of hydrogen and hydroxy;
  • R 3 is selected from the group consisting of hydrogen and -OR 4 ;
  • R 4 is Ci -6 alkyl.
  • R 1 is a member selected from the group consisting of Ci -6 alkoxy, C 0-6 alkoxy-aryl and hydroxy.
  • R 1 is selected from the group consisting of hydroxy, methoxy and benzyloxy.
  • the term aryl is selected from the group consisting of phenyl and naphthyl.
  • the SIRTl inhibitor includes a compound having formula VI:
  • R a is selected from the group consisting of hydrogen, C 1-6 alkyl, aryl,-
  • R b is selected from the group consisting of :
  • X a can be O, S, or NR e ;
  • R c can be hydrogen, C 1-6 alkyl and aryl optionally substituted with a hydrogen, C 1-6 alkyl, aryl, -Ore, -NR e R e , -CN, -NO 2 or halogen;
  • R d can be hydrogen, C 1-6 alkyl, aryl, -Ore, -NR 6 R 6 , or halogen, wherein each instance of R e can be independently hydrogen or C 1-6 alkyl.
  • the SIRTl inhibitor comprising a compound having formula VI has the following structure
  • the disclosure features a cell culture medium that includes a buffered medium, growth factors; and a SIRTl inhibitor having an IC50 for SIRTl enzymatic activity of less than 10 ⁇ M.
  • the SIRTl inhibitor can be, e.g., a SIRTl inhibitor described herein.
  • the disclosure features a cell culture that includes mammalian cells; a medium that contains nutrients, growth factors, and a SIRTl inhibitor having an IC50 for SIRTl enzymatic activity of less than 10 ⁇ M.
  • the SIRTl inhibitor can be, e.g., a SIRTl inhibitor described herein.
  • the packaged product includes a container, a SIRTl inhibitor in the container, and a legend (e.g., a label or insert) associated with the container and indicating how to use the SIRTl inhibitor to culture cells, including any of those cells delineated herein.
  • a legend e.g., a label or insert
  • the disclosure features a method of preparing a donor of cells for transplantation.
  • the method includes administering a SIRTl inhibitor (e.g., an inhibitor described herein) to the donor, and then obtaining cells from the donor for t transplantation.
  • the method can further include transplanting the cells into a recipient.
  • the donor is a mammal, e.g., a pig, a primate, or a human.
  • the donor and the recipient can be the same species, or different species.
  • the disclosure features a method of treating a recipient of transplanted cells.
  • the method includes: administering a SIRTl inhibitor (e.g., an inhibitor described herein) to the recipient, e.g., before, during, or after the recipient receives the transplanted cells.
  • the inhibitor can be provided chronically, e.g., at regular intervals or continuous.
  • the recipient is a mammal, e.g., a pig, a primate, or a human.
  • the disclosure features a SIRTl inhibitor (e.g., an inhibitor described herein) for the enhancement of properties of cells, particularly cultured cells and cells for transplantation.
  • the disclosure features use of a SIRTl inhibitor described herein for the manufacture of a medicament for the enhancement of properties of cells, particularly cultured cells and cells for transplantation.
  • the medicament is administered to an organ donor prior to transplantation, or to a recipient, e.g., before, during, or after transplantation.
  • An exemplary method includes contacting a SIRTl inhibitor to a cell, e.g., a mammalian cell.
  • Cultured cells that have been treated with a SIRTl inhibitor can be transplanted into a subject in need of such a transplant, or can be used in vitro, e.g., to maintain or produce cells for subsequent transplantation, to maintain or produce a cell line of interest, to cryopreserve cells, or to characterize compounds (particularly drugs and drug candidates) for a biological property (e.g., toxicity or cell responsiveness).
  • the methods can be used with a wide variety of cells and with a number of SIRTl inhibitors, for example, as further described below.
  • the cell can be a cell that expresses SIRTl, e.g., a cell that normally expresses SIRTl or that expresses SIRTl as a result of a pathological state.
  • the cells can be of any cell type or lineage, from any tissue, from an adult, infant, fetus, or embryo.
  • the cells can be of any mammalian species.
  • the cells can be pluripotent, multipotent, committed to a cell lineage, differentiating, senescent, quiescent, or terminally differentiated.
  • the cells can be transgenic, e.g., a cell into which a gene has been introduced.
  • the cells can be nullizygous, e.g., in which a gene locus has been disrupted.
  • the cells can be proliferating cells. Further, in some cases, the proliferative capacity of cells may be enhanced or restored (for example, the cells were previously quiescent or senescent) by a SIRTl inhibitor, hi one embodiment, the cells are preferably not transformed or derived from a tumor or cancer sample.
  • the cells can be primary cells obtained from a subject (e.g., a subject (e.g., a mammal, e.g., a human) to whom the cells will be re-introduced, a donor (e.g., a blood donor or biopsy sample)), an established cell line, or cells recently isolated from a subject that have not yet passed through crisis to become an established cell line.
  • a subject e.g., a subject (e.g., a mammal, e.g., a human) to whom the cells will be re-introduced
  • a donor e.g., a blood donor or biopsy sample
  • Cells can be obtained from a subject who is undergoing surgery, e.g., tissue can be removed during surgery; from a blood or plasma sample, e.g., obtained by venous removal with a syringe or from a wound; from a sample obtained by swabbing the interior of a subject's mouth of other orifice; or from a biopsy, e.g., a punch biopsy.
  • Cells, particularly mammalian cells can be selected using a variety of different techniques and settings. Exemplary techniques include flow cytometry sorting (e.g., fluorescence activated cell sorting), antibody-based retention (e.g., on a magnetic bead), and microdissection.
  • Selection techniques include both positive and negative selections or a combination of both techniques. Selections can include one or more repeated steps.
  • a description of positive and negative selection techniques can be found in, for example, U.S. Pat. Nos. 5,925,567, 6,338,942, 6,103,522, 6,117,985, 6,127,135, 6,200,606, 6,342,344, 6,008,040, 5,877,299, 5,814,440, 5,763,266, and 5,677,136.
  • cells of a primary cell line can then be cultured, e.g., as would cells of a primary cell line.
  • the cells can be treated with a SIRTl inhibitor, e.g., by adding the SIRTl inhibitor to the culture medium, e.g., before or after the cells acquire the properties of a primary cell line.
  • primary cell lines are obtained by culturing cells explanted from a subject and treating the cells with a SIRTl inhibitor prior to passage through the crisis point used to create an established cell line.
  • the cells e.g., precursor cells (e.g., stem cells or other progenitor cells), sperm, eggs, cultured cells) can be cryopreserved, e.g., stored under liquid nitrogen, in the presence of a SIRTl inhibitor, for future use (e.g., implantation, transplantation, in vitro culturing or in vitro manipulation, e.g., in vitro fertilization).
  • precursor cells e.g., stem cells or other progenitor cells
  • sperm eggs
  • cultured cells can be cryopreserved, e.g., stored under liquid nitrogen, in the presence of a SIRTl inhibitor, for future use (e.g., implantation, transplantation, in vitro culturing or in vitro manipulation, e.g., in vitro fertilization).
  • the cells are precursor cells, e.g., stem or other progenitor cells.
  • the cells can be obtained, e.g., directly from tissues of an individual, from cell lines, or from less differentiated precursor cells.
  • An exemplary method for obtaining precursor cells from less differentiated cells is described in Gilbert, 1991, Developmental Biology, 3rd Edition, Sinauer Associates, Inc., Sunderland, Mass.
  • the precursor cells can be from any animal, e.g., mammalian, e.g., human, and can be from primary tissue, cell lines, or another source.
  • the precursor cells can be, for example, of ectodermal, mesodermal or endodermal origin.
  • the precursor cell is a stem cell.
  • stem cells include hematopoietic stem cells (HSC; e.g., long term repopulating HSCs), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells, kidney stem cells, and neural stem cells (Stemple and Anderson, Cell 71 :973-985, 1992).
  • HSC hematopoietic stem cells
  • stem cells of epithelial tissues such as the skin and the lining of the gut
  • embryonic heart muscle cells embryonic heart muscle cells
  • liver stem cells liver stem cells
  • kidney stem cells and neural stem cells
  • the stem cells can be expanded in the presence of a SIRTl inhibitor and under conditions that promote proliferation of the cells. Examples of useful HSCs are described in, for example, U.S. Pat. Nos. 5,763,197, 5,750,397, 5,716,827, 5,194,108, 5,061,620, and 4,714,680.
  • Exemplary progenitor cells include pluripotent and multipotent stem cells and stromal cells, and include cells, e.g., stem cells that are committed to a particular cell lineage, e.g., mesenchymal, hematopoietic, adipogenic, hepatogenic, neurogenic, gliogenic, chondrogenic, vasogenic, myogenic, chondrogenic, or osteogenic lineage.
  • stem cells that are committed to a particular cell lineage, e.g., mesenchymal, hematopoietic, adipogenic, hepatogenic, neurogenic, gliogenic, chondrogenic, vasogenic, myogenic, chondrogenic, or osteogenic lineage.
  • stromal cells may be used to promote specific differentiation pathways such as those present in the brain, eye, pharyngeal pancreas, lungs, kidneys, liver, heart, intestine, pancreas, bone, cartilage, skeletal muscle, smooth muscle, ear, esophagus, stomach, blood vessels, and aorta-mesonephros (AGM) region (see e.g., US 2005-0153443).
  • AGM aorta-mesonephros
  • PGC primordial germ cell
  • STO cells fetal fibroblast cells
  • LIF leukaemia inhibitory factor
  • MSCF mouse stem cell factor
  • mice PGCs can also be cultured in similar media to which basic fibroblast growth factor (bFGF) has been added, thereby converting PGCs to cells that resemble undifferentiated embryonic stem cells (ESCs) (Matsui et al., Cell 70:5:841-847, 1992; Resnick et al., Nature 359 (6395):550-551, 1992).
  • bFGF basic fibroblast growth factor
  • PGCs include porcine stem cells . See, e.g., US 6,703,209. Porcine PGCs can be extracted from swine fetuses during about 17 to 39 days post fertilization of the embryo. Preferably, 27 day old fetuses are utilized and PGC suspensions are prepared by trypsin (or EDTA) treatment of genital ridges from the porcine embryos (crossbred) and then seeded on feeder cells (for example in 4-well dishes). The feeder cells are mitotically inactivated and may be STO cells that express MSCF or STO transfected cells (e.g., STO5 or STO8 cells) which express porcine stem cell factor (SCF).
  • SCF porcine stem cell factor
  • cell culture media that may be utilized in addition to such feeder cells or to supplement such feeder cells can be ES medium (Robertson, 1987; Terato-carcinomas and Embryonic Stem Cells, IRL Press) supplemented with 15% FCS; the ES medium supplemented with growth factors such as leukaemia inhibitory factor (LIF) or LIF plus bFGF. Further, a conditioned media prepared from 5637 carcinoma cell lines may be utilized for initial culturing of PGCs.
  • ES medium Robottson, 1987; Terato-carcinomas and Embryonic Stem Cells, IRL Press
  • LIF leukaemia inhibitory factor
  • bFGF bFGF
  • Seeded PGC cultures may be maintained at around 37°C in 5% CO 2 in air.
  • PGCs can be identified by alkaline phosphatase (AP) activity at I 5 3 and 5 days (for example, or at other intervals) using the general procedures set forth above and can also be counted to determine the rate of proliferation.
  • the cultured PGCs are trypsinized, rinsed with fresh medium (such as PBS or ES media) and re-passaged every 5 to 10 days (preferably every 6 or 7 days), but significantly older live cultures obtained from PGCs which have stopped proliferating may be transferred to media containing LIF and PSCF (preferably PSCF is provided by feeder cells) and begin to proliferate.
  • the media used for culturing can include a SIRTl inhibitor, and, for example, porcine SCF and LIF (optionally also including bFGF).
  • Tissue-Specific Cells Cells obtained from a tissue, parts of tissue (e.g., parts of organs or whole-organ cultures), or a progenitor cell type that has been committed to a certain cell lineage or has been stimulated to differentiate to a specific cell type can be cultured by the methods described herein, e.g., in the presence of a SIRTl inhibitor.
  • Tissue cell types include, for example, thymic, lung, liver, brain, muscle, adipocyte, skin, kidney, bone, cartilage, neuronal, gastrointestinal, cardiac, and pancreatic tissue (e.g., pancreatic ⁇ cells).
  • Other cell types include bone marrow cells, cardiac muscle cells, dopamine-producing cells, osteoblasts, osteocytes, hepatocytes, fetal brain cells, or myoblasts.
  • organs include liver, skin, and kidney.
  • the cells can be maintained under a variety of conditions, including, e.g., standard cell/tissue culture techniques, such as those specifically suited for a particular cell type.
  • a SIRTl inhibitor can be added to the cell culture or the SIRTl inhibitor can be combined with culture media prior to the addition of cells, or both.
  • the SIRTl inhibitor can be added once or more than once.
  • SIRTl inhibitor used can vary, e.g., depending on the cell type being cultured (e.g., thymic, lung, liver, brain, muscle, etc.), the status of the cell (e.g., proliferating, quiescent, senescent, pluri-potent, multi-potent, committed to a cell lineage, differentiating, or terminally differentiated).
  • the cell type being cultured e.g., thymic, lung, liver, brain, muscle, etc.
  • the status of the cell e.g., proliferating, quiescent, senescent, pluri-potent, multi-potent, committed to a cell lineage, differentiating, or terminally differentiated.
  • cells can be incubated in humidified chambers at 37°C at 5-15% CO 2 in media containing bulk ions (e.g., Na + , K + , Ca 2+ , Mg 2+ , Cl " , phosphate, bicarbonate or CO 2 ); trace elements (e.g., iron, zinc, selenium); sugars (e.g., glucose); amino acids (e.g., 13 essential amino acids; e.g., L- glutamine); vitamins; choline; inositol; serum (e.g., 5-20% heat-inactivated serum; e.g., that contains growth factors); buffering agents; antibiotics (e.g., streptomycin, amphotericin B, penicillin) to control the growth of bacterial and fungal contaminants.
  • bulk ions e.g., Na + , K + , Ca 2+ , Mg 2+ , Cl " , phosphate, bicarbonate or CO 2
  • trace elements e.g., iron
  • SIRTl inhibitor can be used (e.g., when used for cells that will be transplanted), e.g., in combination (either sequentially or concomitantly) with other agents used to preserve cells, e.g., a cryoprotectant, dimethyl sulfoxide (DMSO) and antioxidants such as glutathione (e.g., reduced glutathione (GSH)), N-acetyl-L-cysteine (NAC), and members of the lazaroid family of 21-aminosteroids (e.g., U-83836E).
  • DMSO dimethyl sulfoxide
  • antioxidants such as glutathione (e.g., reduced glutathione (GSH)), N-acetyl-L-cysteine (NAC), and members of the lazaroid family of 21-aminosteroids (e.g., U-83836E).
  • GSH reduced glutathione
  • NAC N-acetyl-L-cystein
  • the cells that have been contacted with a SIRTl inhibitor can be delivered to a subject, e.g., a subject in need of such cells.
  • the cells are delivered to a subject having cells that are undergoing normal senescence or quiescence.
  • the cells are delivered to a subject that has experienced an injury or a disease, such as heart disease, stroke, kidney failure, liver cirrhosis, or macular degeneration.
  • the cells are delivered to a subject that has experienced a chronically degenerative disease (such as cardiac muscle disease, neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, Huntington's Disease)).
  • a chronically degenerative disease such as cardiac muscle disease, neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, Huntington's Disease)).
  • the cells are delivered to a subject that has experienced a bone disease (e.g., osteoporosis), a blood disease (e.g., a leukemia) or liver disease (e.g., due to alcohol abuse or hepatitis).
  • the cells can be delivered to a subject that has experienced another condition characterized by unwanted cell loss.
  • the cells can be delivered to a subject that has undergone an injury, e.g., chemotherapy and/or radiation treatment.
  • the cells can be delivered to a subject that has suffered a wound, a burn, an ulcer (e.g., ulcer in a diabetic, e.g., diabetic foot ulcer), a surgical wound, a sore, and abrasions.
  • the cells cultured as described herein can be used to repopulate various tissues, such as the liver (Petersen et al., Science 284:1168-1170, 1999) and neuronal tissue (Bjornson et al., Science 283:534-537, 1999).
  • the cells may also serve as a source of cells for various cellular and gene delivery applications.
  • the cells can be used for grafting, e.g., an autograft or allograft (e.g., HSCs can be used for autologous and allogeneic hematopoietic engraftment).
  • the graft can include e.g., soft tissue (e.g., pedicle grafts, free gingival grafts, subepithelial connective tissue grafts), fetal tissue (e.g., fetal brain tissue), nerve tissue, ovarian tissue, bone tissue, connective tissue, corneal tissue, vascularized tissue, and orthopedic grafts.
  • the cells can be used for a xenograft (e.g., graft of tissue from one species to a different species, e.g., from a pig to a human).
  • a xenograft e.g., graft of tissue from one species to a different species, e.g., from a pig to a human.
  • US 6,849,448 describes an exemplary source of cells that is a genetically modified pig, e.g., a pig with an inactivated ⁇ -1,3 galactosyltransferase gene.
  • cells expanded by the methods described herein may be used for gene therapy to treat any of a number of diseases.
  • cells containing a gene that is absent or mutated in the subject or containing a transgene of interest directed toward a particular disease target are prepared in vitro and re-infused into a subject, e.g., such that the cell type(s) targeted by the disease are repopulated by differentiation of the cells in the HSC composition following re-infusion into the subject.
  • the cells can also be genetically modified using gene therapy techniques (see below) to express a desired gene.
  • the modified cells can then be transplanted into a subject, e.g., for the treatment of disease or injury by any method that is appropriate for the type of cells being transplanted and the transplant site.
  • the cells can be transplanted intravenously, or they can be transplanted directly at a target site, e.g., the site of injury or disease.
  • the cells can be maintained in culture or cryopreserved for future studies or transplantation.
  • culturing of a subject's own cells e.g., bone marrow or HSCs (e.g., bone marrow extracted from the subject before commencing chemotherapy or radiation therapy) in the presence of a SIRTl inhibitor can be useful, e.g., to obtain an expanded population of cells and to avoid the current need for immune suppression by minimizing the potential for GVHD following transplantation.
  • a subject's own cells e.g., bone marrow or HSCs (e.g., bone marrow extracted from the subject before commencing chemotherapy or radiation therapy)
  • a SIRTl inhibitor can be useful, e.g., to obtain an expanded population of cells and to avoid the current need for immune suppression by minimizing the potential for GVHD following transplantation.
  • the cells from another source e.g., a donor of the same species (e.g., an HLA-matched donor) or of a different species (e.g, a xenotransplant, e.g., from a transgenic pig)
  • a donor of the same species e.g., an HLA-matched donor
  • a different species e.g., a xenotransplant, e.g., from a transgenic pig
  • the cells cultured by the methods described herein may contain an endogenous gene that is absent from or mutated in cells of the recipient who would receive such cells. These cells can be transplanted to a subject to restore the function provided by the gene that is absent or mutated in the subject.
  • Methods of introduction of cells for transplantation include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and epidural routes.
  • the cells cultured as described herein may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa) and may be administered together with other biologically active agents. Administration can be systemic or local (e.g., into tissue, e.g., liver, muscle, brain, pancreas).
  • the cells cultured as described herein can be derived from the subject to which it is administered, i.e., the transplant is autologous.
  • the cells cultured as described herein can be derived from a heterologous source, e.g., a cell donor (e.g., a blood or platelet donor, a tissue donor, a biopsy sample) or from a primary or established cell line, e.g., a progenitor cell line, e.g., in a pluri-potent or multi-potent state or that has been treated so as to differentiate in a committed cell type lineage.
  • a cell donor e.g., a blood or platelet donor, a tissue donor, a biopsy sample
  • a primary or established cell line e.g., a progenitor cell line, e.g., in a pluri-potent or multi-potent state or that has been treated so as to differentiate in a committed cell type lineage.
  • a progenitor cell line e.g., in a pluri-potent or multi-potent state or that has been treated so as to differentiate in a committed cell type line
  • Such cells can be maintained in culture by the methods described herein to provide a source of cells that can be administered to a subject in the future, or these cells can be used for in vitro studies, e.g., for the characterization of the effects of a compounds or drug on these cells, e.g., the toxicity of a compound and drug or the responsiveness of such cells to a compound or drug.
  • the cells can be cryopreserved for future use.
  • a SIRTl inhibitor can also be administered to a transplant recipient, e.g., a subject who has received transplanted cells, e.g., to maintain or extend the capacity of transplanted precursor cells while in the subject.
  • the SIRTl inhibitor can be provided as a pharmaceutical composition, e.g., subsequent to transplantation, e.g., for a limited or prolonged duration, e.g., for less than two weeks or less than one month.
  • a SIRTl inhibitor can be administered to a transplant donor, e.g., a subject who is donating cells for transplantation (e.g., autologous or heterologous), e.g., to extend or maintain the capacity of the cells to be transplanted prior to donation and/or to maximize the cell harvest obtained from the donor.
  • the SIRTl inhibitor can be provided as a pharmaceutical composition, e.g., prior to transplantation, e.g., for a limited or prolonged duration, e.g., for less than two weeks or less than one month, prior to donation.
  • Gene Delivery Gene delivery encompasses providing an exogenous gene to a cell, e.g., for gene correction therapy and transfer of therapeutic genes, e.g., to treat cancer, infectious diseases, monogenic diseases, multigenic diseases, hereditary diseases, and acquired diseases.
  • the gene can be delivered, e.g., in vitro, e.g., to a cell that has been, is, or will be cultured with a SIRTl inhibitor.
  • Exemplary disease targets include, but are not limited to cancer, such as prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma and leukemia; infectious diseases, such as HIV, monogenic diseases such as CF, hemophilia, phenylketonuria, ADA, familial hypercholesterolemia, and multigenic diseases, such as restenosis, ischemia, and diabetes; degenerative diseases such as neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's Disease. See, e.g., Blaese et al., Science 270:475-480, 1995; Wingo et al., Cancer 82:1197-1207, 1998.
  • infectious diseases such as HIV, monogenic diseases such as CF, hemophilia, phenylketonuria, ADA, familial hypercholesterolemia, and multigenic diseases, such as restenosis, ischemia, and diabetes
  • degenerative diseases such as neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease
  • Cell transduction is possible in vivo. However, it is simpler and more easily controlled ex vivo.
  • a SIRTl inhibitor e.g., such culturing can be performed with HSCs, useful for therapeutic gene therapy (see, e.g., Buetler, Biol. Blood Marrow Transplant. 5:273-276, 1999; Dao, Leukemia 13:1473-1480, 1999; and see generally Morgan et al., Ann. Rev. Biochem. 62:191-217, 1993; Culver et al., Trends Genet. 10:174-178, 1994; and U.S. Pat. No. 5,399,346).
  • a therapeutic gene therapy regimen may include one or more of: obtaining cells from a subject, enriching or purifying the cells of interest, culturing, e.g., expanding, the cells by the methods described herein, introducing the gene of interest into the cells, and reintroducing the cells into the subject.
  • the gene can be contained within a vector.
  • the gene can be introduced, e.g., by transfection or viral transduction.
  • the cells cultured by the methods described herein can be used as recipients for gene delivery.
  • the nucleic acid introduced into the cells may encode any desired protein, e.g., a protein missing or dysfunctional in a disease or disorder.
  • a gene whose expression is desired in a subject can be introduced into the cells such that it is expressible by the cells and/or their progeny, and these cells are then administered in vivo for therapeutic effect.
  • Cells cultured by the methods described herein can be used in any appropriate method of gene therapy.
  • the resulting action of the cells cultured by the methods described herein and carrying a transgene administered to a subject can, for example, lead to the activation or inhibition of a pre-selected gene in the subject, or can provide a gene product that is absent or at low levels in the subject, thus leading to improvement of the diseased condition afflicting the subject.
  • cells cultured by the methods described herein into which a gene has been introduced can be maintained in culture and not transferred to a subject.
  • Such cells can be maintained in culture by the methods described herein to provide a source of cells that can be administered to a subject in the future, or these cells can be used for in vitro studies, e.g., for the characterization of the effects of a compounds or drug on these cells, e.g., the toxicity of a compound and drug or the responsiveness of such cells to a compound or drug.
  • the cells can also be cryopreserved for future use.
  • One common method of practicing gene therapy uses viral vectors, for example retroviral vectors (see Miller et al., Meth.
  • AAV adenovirus-associated vectors
  • adenovirus vectors for example, naked DNA delivered via liposomes, receptor-mediated delivery, calcium phosphate transfection, lipofection, electroporation, particle bombardment (e.g., gene gun), microinjection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, or pressure-mediated gene delivery.
  • particle bombardment e.g., gene gun
  • microinjection cell fusion
  • chromosome-mediated gene transfer e.g., chromosome-mediated gene transfer
  • microcell-mediated gene transfer e.g., spheroplast fusion
  • pressure-mediated gene delivery for example, naked DNA delivered via liposomes, receptor-mediated delivery, calcium phosphate transfection, lipofection, electroporation, particle bombardment (e.g., gene gun), microinjection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, or pressure-mediated gene delivery.
  • the method of transfer can include the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a subject, maintained in culture, or cryopreserved for future use.
  • the technique should provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • a desired gene can also be introduced intracellularly and incorporated within host precursor cell DNA for expression, e.g., by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sd. USA 86:8932-8935, 1989; Zijlstra et al., Nature 342:435-438, 1989).
  • the SIRTl inhibitors can be prepared as purified preparations, e.g., 95%, 96%, 97%, 98%, 99%, or 100% pure.
  • the SIRTl inhibitors can be an active ingredient in a composition (e.g., liquid or solid, e.g., a solid that is added to the cell culture as a solid or is reconstituted (e.g., with a sterile buffer or sterile saline) prior to use.
  • the SIRTl inhibitors can be in a preparation that contains carrier ingredients, e.g., such as salts (e.g., pharmaceutically-acceptable salts), buffers, and/or stabilizers.
  • a particular SIRTl inhibitor can be present in the composition, e.g., between 0.1 — 90% (w/w), e.g., 1-30% (w/w).
  • SIRTl inhibitors include those compounds in one of the three classes described below.
  • One class of compounds that can be used as a SIRTl inhibitor has a general formula (I) and contains a substituted pentacyclic or hexacyclic core containing one or two, respectively, oxygen, nitrogen, or sulfur atoms as a constituent atom of the ring, e.g., X and Y in formula (I) below.
  • R 1 , R 2 , R 3 , and R 4 may include without limitation substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, heteroaryl, etc.
  • the pentacyclic or hexacyclic core may be saturated, i.e. containing no double bonds, or partially or fully saturated, i.e. one or two double bonds respectively.
  • n 0
  • "X" may be oxygen, sulfur, or nitrogen, e.g., NR 7 .
  • the substituent R 7 can be without limitation hydrogen, alkyl, e.g., Cl, C2, C3, C4 alkyl, SO 2 (aryl), acyl, or the ring nitrogen may form part of a carbamate, or urea group.
  • n 1
  • X can be NR 7 , O, or S
  • Y can be NR 7' , O or S.
  • X and Y can be any combination of heteroatoms, e,g,. N,N, N 5 O, N, S, etc.
  • a preferred subset of compounds of formula (I) includes those having one, or preferably, two rings that are fused to the pentacyclic or hexacyclic core, e.g., R 1 and R 2 , together with the carbons to which they are attached, and/or R 3 and R 4 , together with the carbons to which they are attached, can form, e.g., C 5 -C 10 cycloalkyl (e.g., C5, C6, or Cl), C 5 -C 10 heterocyclyl (e.g., C5, C6, or Cl), C 5 -C 10 cycloalkenyl (e.g., C5, C6, or Cl), C 5 -C 10 heterocycloalkenyl (e.g., C5, C6, or Cl), C 6 -C 10 aryl (e.g., C6, C8 or ClO), or C 6 - C 10 heteroaryl (e.g., C5 or C6).
  • Fused ring combinations may include without limitation one or
  • Preferred combinations include B, e.g. having C 6 aryl and C 6 cycloalkenyl (Bl), and C, e.g. having C 6 aryl and C 7 cycloalkenyl (Cl):
  • Each of these fused ring systems may be optionally substituted with substitutents, which may include without limitation halo, hydroxy, C 1 -C 10 alkyl (Cl,C2,C3,C4,C5,C6,C7,C8,C9,C10) , C 1 -C 6 haloalkyl (C1,C2,C3,C4,C5,C6,), C 1 -C 10 alkoxy (Cl,C2,C3,C4,C5,C6,C7,C8,C9,C10).
  • substitutents may include without limitation halo, hydroxy, C 1 -C 10 alkyl (Cl,C2,C3,C4,C5,C6,C7,C8,C9,C10) , C 1 -C 6 haloalkyl (C1,C2,C3,C4,C5,C6,), C 1 -C 10 alkoxy (Cl,C2,C3,C4,C5,C6,C7,C8,C9,C10).
  • C 1 -C 6 haloalkoxy (C1,C2,C3,C4,C5,C6,), C 6 -C 10 aryl (C6,C7,C8,C9,C10), C 5 -C 10 heteroaryl (C5,C6,C7,C8,C9,C10), C 7 -C 12 aralkyl (C7,C8,C9,C1O,C11,C12), C 7 -C 12 heteroaralkyl (C7,C8,C9,C1O,C11,C12), C 3 -C 8 heterocyclyl (C3,C4,C5,C6,C7,C8), C 2 -C 12 alkenyl (C2,C3,C4,C5,C6,C7,C8,C9,C10,Cl l,C12), C 2 -C 12 alkynyl (C2,C3,C4,C5,C6,C7,C8,C9,C10,Cl l,C12), C 5 -C 10 cycloalkenyl (
  • substituents include halo (e.g., fluoro, chloro, bromo), C 1 -C 10 alkyl (e.g., Cl, C2, C3, C4.
  • substitution pattern on the two fused rings may be selected as desired, e.g., one ring may be substituted and the other is not, or both rings may be substituted with 1-5 substitutents (1,2,3,4,5 substitutents).
  • the number of substituents on each ring may be the same or different. Preferred substitution patterns are shown below:
  • the nitrogen substituent R 7 can form a cyclic structure with one of the fused rings containing, e.g., 4-6 carbons, 1-3 nitrogens, 0-2 oxygens and 0-2 sulfurs.
  • This cyclic structure may optionally be substituted with oxo or C 1 -C 6 alkyl.
  • Compounds having activity designated with an A have an IC 50 of less than 1.0 ⁇ M.
  • Compounds having activity designated with a B have an IC 50 between 1.0 ⁇ M and 10.0 ⁇ M.
  • Compounds having activity designated with a C have an IC 50 greater than 10.0 ⁇ M.
  • SIRTl inhibitor compounds that can be used as a SIRTl inhibitor have a general formula (XXIII), (XXIV), (XXV), or (XXVI) and contain a substituted cyclic (e.g., pentacyclic or hexacyclic) or polycyclic core containing one or more oxygen, nitrogen, or sulfur atoms as a constituent atom of the ring(s).
  • a substituted cyclic e.g., pentacyclic or hexacyclic
  • polycyclic core containing one or more oxygen, nitrogen, or sulfur atoms as a constituent atom of the ring(s).
  • formula (XXV) formula (XXVI) Any ring carbon atom can be substituted.
  • the cyclic or polycyclic core may be partially or fully saturated, i.e. one or two double bonds respectively.
  • a preferred subset of compounds of formula (XXIII) includes those having a ring that is fused to the pentacyclic core, e.g., R 1 and R 2 , together with the carbons to which they are attached, and/or R 3 and R 4 , together with the carbons to which they are attached, form C 5 -C 10 cycloalkenyl (e.g., C5, C6, or C7), C 5 -C 10 heterocycloalkenyl (e.g., C5, C6, or Cl), C 6 -C 10 aryl (e.g., C6, CS or ClO), or C 6 -Ci 0 heteroaryl (e.g., C5 or C6).
  • Fused ring combinations may include without limitation one or more of the following:
  • Each of these fused ring systems may be optionally substituted with substitutents, which may include without limitation halo, hydroxy, C 1 -C 10 alkyl (Cl,C2,C3,C4,C5,C6,C7,C8,C9,C10), C 1 -C 6 haloalkyl (C1,C2,C3,C4,C5,C6,), C 1 -C 10 alkoxy (Cl,C2,C3,C4,C5,C6,C7.C8,C9,C10), C 1 -C 6 haloalkoxy (C1,C2.C3,C4,C5,C6,), C 6 -C 10 aryl (C6,C7,C8,C9,C10), C 5 -C 10 heteroaryl (CS 5 CO 5 CT 3 CS 3 CP 5 CIO), C 7 -C 12 aralkyl (C7 3 C8 3 C9 5 C10,Cl l,C12), C 7 -Cj 2 heteroaralkyl (C7,C8,C9,C
  • C2,C3,C4,C5,C6,C7.C8,C9,C10,Cl l,C12 C 5 -C 10 cycloalkenyl (C5 5 C6 5 C7,C8,C9,C10) 5 C 5 -C 10 heterocycloalkenyl (C5,C6,C7 5 C8 5 C9,C10), carboxy, carboxylate, cyano, nitro, amino, C 1 -C 6 alkyl amino (C1 5 C2 3 C3 5 C4 5 C5 5 C6 5 ), C 1 -C 6 dialkyl amino (C1,C2,C3,C4,C5,C6,), mercapto, SO 3 H, sulfate, S(O)NH 2 , S(O) 2 NH 2 , phosphate, C 1 -C 4 alkylenedioxy (C1,C2,C3,C4) 5 oxo, acyl, aminocarbonyl, C 1 -C 6 alkyl aminocarbonyl (C1,C2,C3,C
  • Another preferred subset of compounds of formula (XXIII) includes those where R 1 and R 2 are C 1 -C 6 alkyl (e.g., wherein R 1 and R 2 are both CH 3 ).
  • R 3 is a substituted or unsubstitued aminocarbonyl and R 4 is an amido substituted with a substituent.
  • a preferred subset of compounds of formula (XXIII) 5 X is S.
  • a preferred subset of compounds of formula (XXIV) includes those having a triazole core (i.e., wherein X is NR 16 and both Ys are N).
  • Another preferred subset of compounds include those where R 11 is a substituted thioalkoxy. Where R 11 is thioalkoxy, preferred substituents include aminocarbonyl. An example of a preferred subset is provided below.
  • R 12 is aryl, arylalkyl, heteroaryl, heteroarylalkyl, and alky substituted with heteroaryloxy or aryloxy. Each aryl and heteroaryl is optionally substituted.
  • Still another subset of preferred embodiments include those wherein X is NR 7 and R 7 is aryl, heteroaryl, arylalkyl or heteroarylalkyl, each is which is optionally substituted.
  • a preferred subset of compounds of formula (XXV) includes those having one of the following poly cyclic cores:
  • the polycyclic core can be substituted with one or more suitable substituents.
  • a preferred subset of compounds of formula (XXVI) includes those having the following polycyclic core:
  • the polycyclic core can be substituted with one or more suitable substituents.
  • Other examples of embodiments are depicted in the following structures below together with representative examples of Sir2 activity.
  • Table 2 Activity of Triazoles (cone, in ⁇ M)
  • Compounds having activity designated with an A have an IC 50 of less than 1.0 ⁇ M.
  • Compounds having activity designated with a B have an IC 50 between 1.0 ⁇ M and 10.0 ⁇ M.
  • Compounds having activity designated with a C have an IC 50 greater than 10.0 ⁇ M.
  • Compounds designated with a D were not tested in this assay.
  • the letter X is a member selected from the group consisting of O and S.
  • the symbols L 1 and L 2 each represent members independently selected from the group consisting of O, S, ethylene and propylene, substituted with 0-2 R groups, wherein exactly one of the symbols L and L represents a member selected from the group consisting of O and S.
  • Each instance of the letter R of symbols L 1 and L 2 independently represents a member selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl and- CO 2 R 4 .
  • the symbols R 1 and R 2 each represent members independently selected from the group consisting of hydrogen, C 1-6 alkoxy, Co -6 alkoxy-aryl and hydroxy. Alternatively, the symbols R 1 and R 2 are taken together with the carbons to which they are attached to form a six-membered lactone ring.
  • R 3 represents a member selected from the group consisting of hydrogen, C ⁇ alkyl, aryl,-OR 4 ,-NR 4 R 4 , -CO 2 R 4 , -C(O) R 4 , -C(O)NR 4 R 4 , -CN, -NO 2 and halogen.
  • Each instance of the symbol R 4 independently represents a member selected from the group consisting of hydrogen and Ci- ⁇ alkyl.
  • the compound of Formula V can have the following structure:
  • the symbol R 1 is a member selected from the group consisting of hydrogen, Ci -6 alkoxy and Co -6 alkoxy-aryl ;
  • the symbol R 2 is a member selected from the group consisting of hydrogen and hydroxy;
  • the symbol R 3 is a member selected from the group consisting of hydrogen and -OR 4 ; and
  • the symbol R 4 is Ci- ⁇ alkyl.
  • the symbol R 1 is a member selected from the group consisting of Ci -6 alkoxy, Co -6 alkoxy-aryl and hydroxy.
  • the symbol R 1 is a member selected from the group consisting of hydroxy, methoxy and benzyloxy.
  • the term aryl is a member selected from the group consisting of phenyl and naphthyl.
  • Another exemplary compound has the structure of Formula VI :
  • R a is a member selected from the group consisting of hydrogen, C 1-6 alkyl, aryl,-OR e ,-NR e R e ,-CO 2 R e ,-C(O)R e , -C(O)NR e R e ,-CN,-NO 2 and halogen
  • R b is a member selected from the group consisting of :
  • the symbol X a can be O, S, or NR e .
  • the symbol R c can be hydrogen, Ci -6 alkyl and aryl optionally substituted with a hydrogen, Ci ⁇ alkyl, aryl, -Ore, -NR e R e , -CN, -NO 2 or halogen.
  • the symbol R d can be hydrogen, C h alky., aryl, -Ore, -NR e R e , or halogen.
  • Each instance of the symbol R e can be independently hydrogen or C 1-6 alkyl.
  • a compound of Formula VI has the following structure
  • SIRTl inhibitors include Compound A3 (8,9-dihydroxy-6H- (l)benzofuro[3,2-c]chromen-6-one), Compounds Ml 5 (l-[(4-methoxy-2-nitro- phenylimino)-methyl]-naphthalene-2-ol) and Sirtinol (2-[(2-hydroxy-naphthalen-l- ylmethylene)-amino]-N-(l-phenyl-ethyl)-benzamide).
  • Such compounds are available, e.g., from ChemBridge or can be synthesized. See, e.g., Grozinger et al. J. Biol.
  • SIRTl inhibitors include genes that produce anti- sense nucleic acids that inhibit SIRTl gene expression and other inhibitor agents that can inhibit SIRTl gene expression, e.g., an inhibitor nucleic acid such as an siRNA, anti- sense RNA, or PNA.
  • an inhibitor nucleic acid such as an siRNA, anti- sense RNA, or PNA.
  • Such nucleic acids can be designed to be complementary to a region of the SIRTl mRNA, e.g., near the initiator methionine codon to inhibit translation or expression of the mRNA.
  • the compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention.
  • the compounds of this invention may also contain linkages (e.g., carbon-carbon bonds) or substiruents that can restrict bond rotation , e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis/trans and E/Z isomers are expressly included in the present invention.
  • the compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.
  • compound 3 or 4 can be resolved to a high enantiomeric excess (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 99% or greater) via formation of diasteromeric salts, e.g. with a chiral base, e.g., (+) or (-) ⁇ -methylbenzylamine, or via high performance liquid chromatography using a chiral column.
  • the crude product 4 is purified directly on a chiral column to provide enantiomerically enriched compound. For purposes of illustration, enantiomers of compound 4 are shown below.
  • the compounds disclosed herein are administered where one isomer (e.g., the R isomer or S isomer) is present in high enantiomeric excess.
  • an isomer of compounds 6 or 7 that has a greater affinity for SirTl than its enantiomer (i.e., an enantiomerically enriched preparation).
  • a compound having the one of the following structures where the stereochemical structure of the amide (or other substituent) corresponds to the amide in compound 4 having a negative optical rotation i.e., an enantiomeric enriched compound.
  • Salts of the SIRTl inhibitors include those derived from inorganic and organic acids and bases.
  • suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate
  • Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts. It is also possible to have the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil- soluble or dispersible products may be obtained by such quaternization. Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxy groups (e.g. L-arginine, -lysine, -histidine salts).
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the cell being cultured and other culture conditions.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • such preparations contain from about 20% to about 80% active compound. Lower or higher doses than those recited above may be required.
  • the ability of a compound to inhibit SIRTl can be evaluated in many ways.
  • interaction with, e.g.,. binding of, SIRTl can be assayed in vitro.
  • the reaction mixture can include a SIRTl co-factor such as NAD and/or a NAD analog.
  • the ability of the compound to inhibit an enzymatic function, e.g., deacetylase activity of SIRTl can be assayed in vitro.
  • Assays can include determining the IC50 of the compound.
  • An exemplary assay method includes a multi-well format of the SirTl enzymatic assay that is based on the commercial "Fluor-de-Lys" assay principle by Biomol, which is fluorogenic (www.biomol.com/store/Product_Data_PDFs/ak500.pdf).
  • deacetylation of the ⁇ -amino function of a lysyl residue is coupled to a fluorogenic development step that is dependent on the unblocked ⁇ -amino functionality and generates fluorescent aminomethylcoumarin. Fluorescence can be read on a commercial macroscopic reader. Standard enzymological analyses can be used to determine K;.
  • the assay includes contacting the SIRTl protein or biologically active portion thereof with a known compound which binds a SIRTl to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a SIRTl protein, wherein determining the ability of the test compound to interact with the SIRTl protein includes determining the ability of the test compound to preferentially bind to the SIRTl or biologically active
  • cell free assays can be conducted in a liquid phase.
  • the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation
  • fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.
  • Sirtuins are further described, and additional exemplary sequences are presented in U.S. Patent Application No. 11/018,018 (filed on December 20, 2004).
  • Sirtuins are members of the Silent Information Regulator (SIR) family of genes.
  • SIRTl proteins bind to a number of other proteins, referred to as "SIRTl binding partners.”
  • SIRTl binds to p53 and plays a role in the p53 pathway, e.g., K370, K371, K372, K381, and/or K382 of p53 or a peptide that include one or more of these lysines.
  • the peptide can be between 5 and 15 amino acids in length.
  • SIRTl proteins can also deacetylate histones.
  • SIRTl can deacetylate lysines 9 or 14 of histone H3 or small peptides that include one or more of these lysines.
  • Histone deacetylation alters local chromatin structure and consequently can regulate the transcription of a gene in that vicinity.
  • Many of the SIRTl binding partners are transcription factors, e.g., proteins that recognize specific DNA sites. Interaction between SIRTl and SIRTl binding partners can deliver SIRTl to specific regions of a genome and can cause local changes to the acetylation of substrates, e.g., histones and transcription factors localized to the specific region. It has been found that a genetic deficiency of SIRTl in mouse embryonic fibroblasts dramatically increases resistance to replicative senescence (Chua et al., Cell Metabolism 2:67, 2005).
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3- phenylpropionate, phosphate,
  • alkali metal e.g., sodium
  • alkaline earth metal e.g., magnesium
  • ammonium e.g., sodium
  • N-(alkyl) 4 salts e.g., sodium
  • This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.
  • Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxy groups (e.g., o i L-arginine, -lysine, -histidine salts).
  • the compounds of the formulae described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.5 to
  • compositions of this invention will be administered from about 1 to about
  • Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that maybe combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • active compound w/w
  • 5 preparations contain from about 20% to about 80% active compound.
  • compositions delineated herein include the compounds of the formulae delineated herein, as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms, including those described herein.
  • pharmaceutically acceptable carrier or adjuvant refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- ⁇ -tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-
  • Cyclodextrins such as ⁇ -, ⁇ -, and ⁇ -cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl- ⁇ -cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.
  • the pharmaceutical compositions of this invention maybe administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection.
  • the pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions, m the case of tablets for oral use, carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • the active ingredient When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
  • compositions of this invention may also be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • Topical administration of the pharmaceutical compositions of this invention is useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
  • the pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • a composition having the compound of the formulae herein and an additional agent can be administered using an implantable device.
  • Implantable devices and related technology are known in the art and are useful as delivery systems where a continuous, or timed-release delivery of compounds or compositions delineated herein is desired. Additionally, the implantable device delivery system is useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs). Negrin et al., Biomaterials, 22(6):563 (2001). Timed-release technology involving alternate delivery methods can also be used in this invention. For example, timed-release formulations based on polymer technologies, sustained-release techniques and encapsulation techniques (e.g., polymeric, liposomal) can also be used for delivery of the compounds and compositions delineated herein.
  • a patch to deliver active chemotherapeutic combinations herein.
  • a patch includes a material layer (e.g., polymeric, cloth, gauze, bandage) and the compound of the formulae herein as delineated herein.
  • One side of the material layer can have a protective layer adhered to it to resist passage of the compounds or compositions.
  • the patch can additionally include an adhesive to hold the patch in place on a subject.
  • An adhesive is a composition, including those of either natural or synthetic origin, that when contacted with the skin of a subject, temporarily adheres to the skin. It can be water resistant. The adhesive can be placed on the patch to hold it in contact with the skin of the subject for an extended period of time.
  • the adhesive can be made of a tackiness, or adhesive strength, such that it holds the device in place subject to incidental contact, however, upon an affirmative act (e.g., ripping, peeling, or other intentional removal) the adhesive gives way to the external pressure placed on the device or the adhesive itself, and allows for breaking of the adhesion contact.
  • the adhesive can be pressure sensitive, that is, it can allow for positioning of the adhesive (and the device to be adhered to the skin) against the skin by the application of pressure (e.g., pushing, rubbing,) on the adhesive or device.
  • compositions of this invention comprise a combination of a compound of the formulae described herein and one or more additional therapeutic or prophylactic agents
  • both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
  • the additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition.

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Abstract

L'invention concerne une méthode de mise en culture de cellules en présence d'un inhibiteur de SIRT1.
PCT/US2006/040426 2005-10-18 2006-10-17 Inhibition de sirt1 Ceased WO2007047604A2 (fr)

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WO2008011557A3 (fr) * 2006-07-20 2008-07-31 Allen J Borchardt Inhibiteurs hétéroaryliques de la kinase rho
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WO2017120409A1 (fr) * 2016-01-06 2017-07-13 Auspex Pharmaceuticals, Inc. Inhibiteurs tétrahydrocarbazole de récepteurs sirt1
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US7968587B2 (en) 2006-11-20 2011-06-28 Eli Lilly And Company Tetrahydrocyclopenta[b]indole compounds as androgen receptor modulators
US20140030295A1 (en) * 2008-02-07 2014-01-30 The J. David Gladstone Institutes Use of Sirt1 Activators or Inhibitors to Modulate an Immune Response
US8486943B2 (en) 2008-05-16 2013-07-16 Eli Lilly And Company Tetrahydrocyclopenta[b]indole androgen receptor modulators
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JP2012513481A (ja) * 2008-12-23 2012-06-14 プレジデント アンド フェロウズ オブ ハーバード カレッジ ネクロトーシスの小分子阻害剤
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WO2012155209A1 (fr) * 2011-05-19 2012-11-22 Mesoblast, Inc Méthodes de traitement de l'obésité et/ou du syndrome métabolique
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US9676760B2 (en) 2011-07-01 2017-06-13 Gilead Sciences, Inc. Fused heterocyclic compounds as ion channel modulators
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US20140303382A1 (en) * 2011-10-20 2014-10-09 Siena Biotech S.P.A. Process for the preparation of 6-chloro-2,3,4,9-tetrahydro-1h-carbazole-1-carboxamide and intermediates thereof
US9725452B2 (en) 2013-03-15 2017-08-08 Presidents And Fellows Of Harvard College Substituted indoles and pyrroles as RIP kinase inhibitors
US10017520B2 (en) 2014-12-10 2018-07-10 Massachusetts Institute Of Technology Myc modulators and uses thereof
US9884819B2 (en) 2016-01-06 2018-02-06 Auspex Pharmaceuticals, Inc. Tetrahydrocarbazole inhibitors of SIRT1 receptors
WO2017120409A1 (fr) * 2016-01-06 2017-07-13 Auspex Pharmaceuticals, Inc. Inhibiteurs tétrahydrocarbazole de récepteurs sirt1
US10106555B2 (en) 2016-02-16 2018-10-23 Massachusetts Institute Of Technology Max binders as MYC modulators and uses thereof
US10865213B2 (en) 2016-02-16 2020-12-15 Massachusetts Institute Of Technology Max binders as MYC modulators and uses thereof
US11542278B1 (en) 2020-05-05 2023-01-03 Nuvalent, Inc. Heteroaromatic macrocyclic ether chemotherapeutic agents
US11667649B2 (en) 2020-05-05 2023-06-06 Nuvalent, Inc. Heteroaromatic macrocyclic ether chemotherapeutic agents
US12054498B2 (en) 2020-05-05 2024-08-06 Nuvalent, Inc. Heteroaromatic macrocyclic ether chemotherapeutic agents
US12275742B2 (en) 2020-05-05 2025-04-15 Nuvalent, Inc. Heteroaromatic macrocyclic ether chemotherapeutic agents
US12043626B2 (en) 2021-10-01 2024-07-23 Nuvalent, Inc. Solid forms, pharmaceutical compositions and preparation of heteroaromatic macrocyclic ether compounds
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